Abstract
Akebia quinata is an over-harvesting shrub which faces an urgent need for reasonable conservation strategies. In this study, we report its complete chloroplast genome by Illumina pair-end sequencing. The total cp genome size was 157,817 bp in length, containing a pair of inverted repeats of 26,143 bp, separated by large single copy and small single copy of 86,543 bp and 18,988 bp, respectively. The chloroplast genome of A. quinata encodes 113 different genes, including 79 protein-coding genes, 30 transfer RNAs and 4 ribosomal RNAs. A total of 47 perfect cp microsatellites were analyzed in the A. quinata. The majority of the SSRs in this cp genome are mononucleotides (74.47 %). Regions of the highest variability were sought out by comparing with A. trifoliate. There are only 385 nucleotide substitutions and 141 indels between the two genomes. Six highly variable regions were identified including four intergenic regions and two coding regions.
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The stems of Akebia plants, Akebiae Caulis, is an herbal medicine that has long been widely used as an antiphlogistic, a diuretic, and an analgesic in traditional oriental medicine (Kitaoka et al. 2009). Akebia quinata was distributed in southeast China around the Yangtze River. Wild resources of this species have been seriously deteriorated due to years of over-harvesting, indicating an urgent need for reasonable conservation strategies (Li et al. 2010). A good knowledge of genomic information of this genus can provide insights for conservation and restoration efforts.
Because of their highly conserved structure, uniparental inheritance, haploid and general recombination-free quality, chloroplast genomes provide valuable information for plant species conservation, DNA barcoding, taxonomy and phylogeny (Dong et al. 2013b, 2014). Here, we sequenced and analyzed the chloroplast genome of A. quinata based on the next-generation sequencing method (Dong et al. 2013a). Our aim was to retrieve valuable cp molecular markers, indels and SSRs for this genus or species, by comparing the cp genomes with the recently published cp genome of A. trifoliate (Sun et al. 2016).
Total Genomic DNA was extracted from fresh young leaves of an A. quinata plant found in the Sangzhi, Zhangjiajie, Hunan province (collection number: libin-02) using the mCTAB method (Li et al. 2013). DNA was sheared to construct 400 bp (insert size) paired-end library in accordance with the Illumina HiSeq Xten platform. Approximately 6.1 Gb of raw data were generated with paired-end 150 bp read length. The paried-end reads were qualitatively assessed and assembled with SPAdes 3.6.1 (Bankevich et al. 2012). The annotation was performed with Plann (Huang and Cronk 2015). The circular cp genome maps were drawn using the OGDRAW program (Lohse et al. 2013). The annotated genomic sequence had been submitted to GenBank with the accession number KX611091.
The size of the cp genome of A. quinata is 157,817 bp. The cp genome exhibits a quadripartite structure, which includes a pair of inverted repeats (IRa and IRb 26,143 bp), and separated SSC (18,988 bp) and LSC (86,543) regions (Fig. 1). The GC content of the chloroplast DNA is 38.7 %. The GC content of the LSC (37.1) and SSC regions (33.7) are lower than that of the IR regions (43.1 %). The chloroplast genome of A. quinata encodes 113 different genes, including 79 protein-coding genes, 30 transfer RNAs (tRNA) and 4 ribosomal RNAs (rRNA). Eighteen genes are duplicated in the IR, including seven protein-coding genes, seven tRNA genes and four rRNA genes. Fifteen distinct genes contain a single intron while ycf3 and clpP each contains two.
Microsatellites (mono-, di-, tri-, tetra-, penta-, and hexanucleotide repeats) were detected using the Perl script MISA (MIcroSAtellite; http://pgrc.ipk-gatersleben.de/misa). The minimum numbers (thresholds) of repeats were 10, 5, 4, 3, 3 and 3 for mono-, di-, tri-, tetra-, penta-, and hexanucleotides, respectively. A total of 47 perfect cp microsatellites were analyzed in the A. quinata. The majority of the SSRs in this cp genome are mononucleotides (74.47 %) and almost all of the mononucleotides (97.14 %) are composed of A/T. Furthermore, di-, tri-, tetra-nucleotide repeats each had four numbers.
Two Akebia cp genome sequences were aligned using MAFFT v7 (Katoh and Standley 2013) and adjusted manually. Furthermore, a sliding window analysis was conducted for variability (π) evaluation using the DnaSP v5 software (Librado and Rozas 2009). The step size was set to 200 bp, with a 600-bp window length. A comparison of the entire chloroplast genome sequences of A. quinata and A. trifoliate revealed 385 nucleotide substitutions and 141 indels. The variability throughout the chloroplast genomes was quantified using the average nucleotide diversity (π) (Fig. 2). The average value of π is 0.00245. There were six peaks which showed remarkably higher π values (>0.01). Four of the peaks are intergenic regions of trnH-psbA, trnK-matK, petA-psbJ, and rpl32-trnL, the other two are the coding regions of ccsA and ndhD (Fig. 2). In all, these findings provide a valuable analysis of the complete cp genome of A. quinata, which adds to our understanding of Akebia genetic resources and will facilitate the Akebia species identification, phylogeny analysis, and conservation genetics.
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Acknowledgments
This work was supported by the National Forest Genetic Resources Platform 2015. The authors thank Wenpan Dong and Chao Xu for their help and assistance in sequencing chloroplast genome.
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Li, B., Li, Y., Cai, Q. et al. Development of chloroplast genomic resources for Akebia quinata (Lardizabalaceae). Conservation Genet Resour 8, 447–449 (2016). https://doi.org/10.1007/s12686-016-0593-0
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DOI: https://doi.org/10.1007/s12686-016-0593-0